Oxidative stress has been proposed to be a major cause of many neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). In these and other conditions, neuronal damage or death can be caused by reactive oxygen and nitrogen species, which are generated by endothelial cells as well as astrocytes and glia, reacting with nearby neurons. Cytokines and other peptides released by astrocytes during infection or inflammation cause the induction of inducible nitric oxide synthase (iNOS), which results in the release of large amounts of nitric oxide into the neurovascular space. This nitric oxide can react with superoxide generated inside neurons or other cells to produce peroxynitrite as well as other reactive oxygen and nitrogen species. The overall goal of this proposal is to develop MEMs-based analytical methodology that will enable researchers to obtain quantitative information regarding the concentration of reactive nitrogen species generated by the cells that make up the neurovascular space. Microchip-based devices provide some unique advantages for these studies. The small dimensions of the channels on a microchip make it possible to measure samples with very small volumes (including the picoliter volumes contained in a single cell). The integrated format of the chip makes it possible to detect short-lived species, such as peroxynitrite, as they are formed by chemical, enzymatic, or biological reactions. Fast and efficient separations of these transient species can be accomplished in less than a minute by microchip electrophoresis. In addition, detectors can be directly integrated into the chip format for the selective and sensitive detection of redox-active or fluorescent analytes. Our plan for this proposal is to develop microchip-based systems for the detection of peroxynitrite and other reactive oxygen and nitrogen species. This methodology will be used to monitor the production of peroxynitrite and its reaction products both in chemical reactions and in biological systems. Initial studies will concentrate on the detection of peroxynitrite generated by macrophages because they are known to produce large quantities of this compound upon activation. However, the ultimate goal will be to measure the peroxynitrite generated by astrocytes and endothelial cells present in the neurovascular space and at the blood-brain barrier. Analyses will be performed both at the single cell level and with live cells in culture. This methodology can then be used to obtain a better understanding of the role of oxidative stress in the development, progression, and regulation of neurodegenerative diseases. PUBLIC HEALTH RELEVANCE: This proposal is concerned with the development of an analytical method for the measurement of peroxynitrite in living systems. Peroxynitrite is a free radical species that is generated in the brain during conditions of oxidative stress and is believed to play a major role in neurodegenerative diseases including Alzheimer's and Parkinson's disease. The proposed methodology will make it possible to better elucidate the role of this extremely short lived species in neurodegenerative disease.